OBJECTIVE:: To estimate whether African ancestry, specific gene polymorphisms, and gene-environment interactions could account for some of the unexplained preterm birth variance within African American women. METHODS:: We genotyped 1,509 African ancestry-informative markers, cytochrome P450 1A1 (CYP1A1), and glutathione S-transferases Theta 1 (GSTT1) variants in 1,030 self-reported African American mothers. We estimated the African ancestral proportion using the ancestry-informative markers for all 1,030 self-reported African American mothers. We examined the effect of African ancestry and CYP1A1- and GSTT1-smoking interactions on preterm birth cases as a whole and within its subgroups: very preterm birth (gestational age less than 34 weeks); and late preterm birth (gestational age greater than 34 and less than 37 weeks). We applied logistic regression and receiver operating characteristic curve analysis, separately, to evaluate whether African ancestry and CYP1A1- and GSTT1-smoking interactions could make additional contributions to preterm birth beyond epidemiologic factors. RESULTS:: We found significant associations of African ancestry with preterm birth (22% compared with 31%, odds ratio [OR] 1.11, 95% confidence interval [CI] 1.02-1.20) and very preterm birth (23% compared with 33%, OR 1.17, 95% CI 1.03-1.33), but not with late preterm birth (22% compared with 29%, OR 1.06, 95% CI 0.97-1.16). In addition, the receiver operating characteristic curve analysis suggested that African ancestry and CYP1A1- and GSTT1-smoking interactions made substantial contributions to very preterm birth beyond epidemiologic factors. CONCLUSION:: Our data underscore the importance of simultaneously considering epidemiologic factors, African ancestry, specific gene polymorphisms, and gene-environment interactions to better understand preterm birth racial disparity and to improve our ability to predict preterm birth, especially very preterm birth. LEVEL OF EVIDENCE:: II.